Expression of circular RNA circ_0007823 in triple negative breast cancer and its impact on cellular biological behaviors

doi:10.3877/cma.j.issn.1674-0807.2022.04.002

Abstract

Abstract:

Objective

To investigate the expression of circular RNA circ_0007823 in triple negative breast cancer (TNBC) and potential mechanism of circ_0007823 affecting the biological behaviors of TNBC cells.

Methods

Using random number table, 30 cases were selected from 120 female cases with TNBC who met the inclusion and exclusion criteria in the Changning Maternal and Child Health Hospital, Shanghai from January 2016 to December 2020 for a retrospective study. The expression of circ_0007823 in TNBC tissue and adjacent normal tissue was detected using real-time quantitative RT-PCR and compared by t test. With the median circ_0007823 expression in TNBC tissue as the cutoff value, 30 cases were divided into high expression group and low expression group and the clinicopathological characteristics were compared between two groups using Fisher’s exact test. In order to explore the role of circ_0007823 overexpression on TNBC cells, MDA-MB-231 cells were transfected with circ_0007823 overexpression plasmid and empty plasmid, respectively (overexpression group and control group). At 48 h after transfection, the migration and invasion abilities of cells were detected by Transwell assay, and the proliferation of cells was detected by CCK-8 method. To further investigate the role of circ_0007823 mutation in TNBC cells, MDA-MB-231 cells were transfected with empty, circ_NC mutant and circ_NC wild-type plasmids, respectively (circ_NC group, circ_0007823-Mut group and circ_0007823-WT group). At 48 h after transfection, the migration and invasion abilities of cells were detected by Transwell assay; at 0, 24, 48, 72 and 96 h after transfection, the proliferation of cells were detected by CCK-8 method. The relationship between circ_0007823 and miR-182-5p was verified by the dual-luciferase reporter assay. RT-PCR and Western blot analysis were used to detect the expression of miR-182-5p and target gene FOXF2 in MDA-MB-231 cells with overexpression of circ_0007823. t test was used to compare the number of migrating cells and invasive cells between two groups, one-way analysis of variance with Tukey correction was used for multiple comparison. The optical density of cells was compared between two groups using t test, repeated measures analysis of variance was used for comparison among multiple groups at different time points and Bonferroni correction was used for pairwise comparison.

Results

The expression of circ_0007823 in TNBC tissue was significantly lower than that in adjacent normal tissue (5.97±3.08 vs 20.28±5.44, t=16.469, P<0.001). The expression of circ_0007823 in TNBC tissue was correlated with lymph node metastasis (P=0.014). Compared with control group, a significant decrease was observed in the number of migrating cells, number of invasive cells and optical density of cells in circ_0007823 overexpression group (120.67±6.81 vs 107.00±3.61, t=5.857, P=0.028; 94.00±3.61 vs 58.33±3.06, t=107.000, P<0.001; 0.73 ± 0.02 vs 0.63 ± 0.02, t=7.187, P=0.001). The number of migrating cells in the circ_NC group, circ_0007823-Mut group, and circ_0007823-WT group was 120.67±6.81, 109.67±6.51, 82.00±17.91, respectively, indicating a significant difference (F=38.550, P<0.001); the numbers of invasive cells was 272.33±6.03, 261.67±2.52, 194.67±9.02, respectively, indicating a significant difference (F=128.648, P<0.001). The number of migrating and invasive cells in circ_0007823-WT group was significantly lower than that in control group and circ_0007823-Mut group (all P<0.001). The optical density of MDA-MB-231 cells in the circ_NC group, circ_0007823-Mut group and circ_0007823-WT group indicated a significant difference (comparison between groups, F=297.883, P<0.001; comparison among different time points, F=1 207.174, P<0.001; interaction, F=42.433, P<0.001). The result of dual-luciferase reporter assay confirmed that circ_0007823 had targeted binding sites with miR-182-5p. Compared with control group, after transfection with circ_0007823 overexpression plasmid in MDA-MB-231 cells, the expression of circ_0007823 increased (1.01±0.9 vs 9.30±2.47, t=-5.794, P=0.029), the expression of miR-182-5p decreased (1.00±0.11 vs 0.86±0.09, t=2.838, P=0.022), and the mRNA expression of FOXF2 increased (0.87±0.11 vs 1.06±0.24, t=2.697, P=0.027).

Conclusion

circ_0007823, which is lowly expressed in TNBC tissue, has inhibitory effect on the proliferation, invasion and migration of TNBC cells and its targeted binding with miR-182-5p can up-regulate the expression of FOXF2.

Key words: Triple negative breast cancer, Migration, MicroRNAs, circ_0007823

Jinling Yu, Beimin Gao, Jing Cui, Haofeng Wang, Jiangfan Zhu, Weida Shen. Expression of circular RNA circ_0007823 in triple negative breast cancer and its impact on cellular biological behaviors[J]. Chinese Journal of Breast Disease(Electronic Edition), 2022, 16(04): 204-211.

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References 17

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引物名称	引物序列
miR-182-5p-RT	5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTGTG-3′
miR-182-5p-hF	5′-GCCTTTGGCAATGGTAGAACT-3′
FOXF2-hF	5′-CCGTTACCAGCATCACTCTACT-3′
FOXF2-hR	5′-CGCAGGGCTTAATATCCTGACA-3′
circ-0007823-hF	5′-CTCCAGAATTTGGGCAATA-3′
circ-0007823-hR	5′-AACCTGCATCCATGTCTCC-3′
U6-hF	5′-CTCGCTTCGGCAGCACA-3′
U6-hR	5′-AACGCTTCACGAATTTGCGT-3′
U6-RT	5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATG-3′
GAPDH-hF	5′-TGACAACTTTGGTATCGTGGAAGG-3′
GAPDH-hR	5′-AGGCAGGGATGATGTTCTGGAGAG-3′

引物名称	引物序列
miR-182-5p-RT	5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTGTG-3′
miR-182-5p-hF	5′-GCCTTTGGCAATGGTAGAACT-3′
FOXF2-hF	5′-CCGTTACCAGCATCACTCTACT-3′
FOXF2-hR	5′-CGCAGGGCTTAATATCCTGACA-3′
circ-0007823-hF	5′-CTCCAGAATTTGGGCAATA-3′
circ-0007823-hR	5′-AACCTGCATCCATGTCTCC-3′
U6-hF	5′-CTCGCTTCGGCAGCACA-3′
U6-hR	5′-AACGCTTCACGAATTTGCGT-3′
U6-RT	5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATG-3′
GAPDH-hF	5′-TGACAACTTTGGTATCGTGGAAGG-3′
GAPDH-hR	5′-AGGCAGGGATGATGTTCTGGAGAG-3′

临床病理特征		例数	circ_0007823表达		P值
临床病理特征		例数	低表达(n＝15)	高表达(n＝15)	P值
年龄
	≤50岁	15	9	6	0.466
	>50岁	15	6	9	0.466
绝经状态
	否	13	7	6	0.713
	是	17	8	9	0.713
淋巴结转移
	否	21	7	14	0.014
	是	9	8	1	0.014
组织学分级
	2	12	7	5	0.456
	3	18	8	10	0.456
TNM分期
	Ⅰ+Ⅱ	26	11	15	0.100
	Ⅲ+Ⅳ	4	4	0	0.100
Ki-67表达
	≥20%	7	3	4	0.666
	<20%	23	12	11	0.666

临床病理特征		例数	circ_0007823表达		P值
临床病理特征		例数	低表达(n＝15)	高表达(n＝15)	P值
年龄
	≤50岁	15	9	6	0.466
	>50岁	15	6	9	0.466
绝经状态
	否	13	7	6	0.713
	是	17	8	9	0.713
淋巴结转移
	否	21	7	14	0.014
	是	9	8	1	0.014
组织学分级
	2	12	7	5	0.456
	3	18	8	10	0.456
TNM分期
	Ⅰ+Ⅱ	26	11	15	0.100
	Ⅲ+Ⅳ	4	4	0	0.100
Ki-67表达
	≥20%	7	3	4	0.666
	<20%	23	12	11	0.666

组别	样本量	吸光度
组别	样本量	0 h	24 h	48 h	72 h	96 h
circ_NC组	30	0.89±0.02	1.00±0.00^a	1.74±0.01^a	2.08±0.03^a	2.11±0.05^a
circ_0007823-WT组	30	0.87±0.01	0.89±0.01	1.36±0.01	1.44±0.01	1.47±0.01
circ_0007823-Mut组	30	0.86±0.03	0.95±0.02	1.64±0.06	1.96±0.02^a	1.99±0.01^a

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